Project Summary Notch receptors are mechanically activated transmembrane proteins that play important roles in regulating cell fate, differentiation, proliferation, adhesion, and many other critical processes. However, the majority of studies to date have been focused largely on understanding Notch from biochemical and genetic perspectives, and only recently been explored as a mechanoreceptor. Given the varied roles Notch plays in both normal and pathological states, it is necessary to formulate an integrated mechano-chemical perspective of Notch signaling and regulation as such outlook is required to achieve a comprehensive view of this critical pathway. In this project, my lab will leverage our expertise in chemical probe development and molecular tool design in order to address sharply focused mechanistic questions regarding Notch mechanotransduction. In order to distinguish our contributions from those of others, we will pursue a multi-scale understanding of the pathway by combining single molecule studies and high-resolution imaging with the aim of understanding how the activation of the receptor is coupled to biomechanical events that occur within the cell. In particular, new correlative light and electron microscopy will be developed and applied to determine the precise timing and location of events surrounding Notch signal transduction. In addition, in order to address important questions regarding the source and magnitude of the external forces that are experienced by cells, we will also create synthetic versions of Notch and apply them as genetically encoded ?tensiometers.? Successful execution of this work will provide deep insights into the synergy occurring between biochemical and mechanical cues as well as increase our overall understanding of the how cells sense and interpret mechanical information.